EP0839198A1 - Vecteur et systeme d'ancrage superficiel pour proteines etrangeres - Google Patents

Vecteur et systeme d'ancrage superficiel pour proteines etrangeres

Info

Publication number
EP0839198A1
EP0839198A1 EP97915739A EP97915739A EP0839198A1 EP 0839198 A1 EP0839198 A1 EP 0839198A1 EP 97915739 A EP97915739 A EP 97915739A EP 97915739 A EP97915739 A EP 97915739A EP 0839198 A1 EP0839198 A1 EP 0839198A1
Authority
EP
European Patent Office
Prior art keywords
inp
vector
recombinant vector
gene
foreign
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97915739A
Other languages
German (de)
English (en)
Inventor
Jae Gu Pan
Heung Chae Jung
Seung Hwan Park
Moon Hi Han
Young Hoon Park
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Korea Advanced Institute of Science and Technology KAIST
Korea Institute of Science and Technology KIST
Original Assignee
Korea Advanced Institute of Science and Technology KAIST
Korea Institute of Science and Technology KIST
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Korea Advanced Institute of Science and Technology KAIST, Korea Institute of Science and Technology KIST filed Critical Korea Advanced Institute of Science and Technology KAIST
Publication of EP0839198A1 publication Critical patent/EP0839198A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the present invention relates to surface anchoring vectors containing gene segments of ice nucleation protein (INP) , which can express foreign proteins onto cell surface. And the present invention relates to a method for preparing foreign proteins onto cell surface by using INP and use of foreign proteins prepared by the surface expression system.
  • INP ice nucleation protein
  • the present invention relates to surface anchoring vectors, a method for preparing foreign proteins onto cell surface and use thereof, which uses outer cell membrane proteins, ice nucleation protein
  • proteins should pass through inner cell membrane and periplasmic space, localize onto outer cell membrane stably and be extruded.
  • surface proteins, specific enzymes and toxin proteins have a secretion signal and a targeting signal localizing the proteins onto cell surface.
  • foreign proteins can be expressed onto the cell surface successfully by using such a secretion signal, a targeting signal and the like combined with proper promoters.
  • surface proteins present in gram-negative bacteria have been utilized mainly to produce foreign polypeptides which is necessary onto cell surface.
  • proteins used for the cell surface expression such as outer cell membrane protein, lipoprotein, secretion protein and cell surface structure protein.
  • outer cell membrane proteins Lam B, Pho E, Omp A and the like have been utilized for the surface expression.
  • sizes of proteins expressed onto cell surface is limited since the proteins should be inserted into loops extruded from the cell surface.
  • the C- and N-termini of the foreign protein should be close to each other 3- di ensionally. Thus, when the distance between the two termini is long the C- and N-termini should be joined.
  • Lam B or Pho E is utilized to insert foreign polypeptides comprising more than 50-60 amino acids, membrane proteins can not be produced stably due to structural limitation [Charbit, et al., J. Immunol., 139: 1658-1664 (1987); Agterberg, et al. , Vaccines, 8: 85-91 (1990)] .
  • structural limitation a part of Omp A protein which contains minimum targeting sequence localizing foreign proteins onto outer cell membrane is used, although the whole Omp A protein was also used to insert foreign proteins into extruded loop.
  • ⁇ -lactamase was expressed onto the cell surface by fusing the C-terminal targeting sequence of Omp A protein.
  • Omp A fragment helped proteins expressed and bound onto cell surface and a signal sequence of E. coli lipoprotein, Lpp, which was fused to N-terminus of Omp A helped the protein localized onto outer cell membrane [Francisco, et al. , Proc. Natl. Acad. Sci. USA, 489: 2713-2717 (1992)1.
  • outer membrane proteins selected as a surface anchoring motif must satisfy requirements described below.
  • the outer membrane proteins should i) have a secretion signal by which fusion protein can pass through inner cell membrane, ii) have a targeting signal by which the protein can bind onto outer cell membrane, iii) be expressed massively on the cell surface and iv) be expressed stably regardless of protein size.
  • any cell surface anchoring motif satisfying all the requirements has not been yet developed and only supplemented the above disadvantages.
  • Lipoproteins as a surface protein, has been also used for the surface expression.
  • E. coli lipoproteins can pass through inner cell membrane due to secretion signal at the N-terminus and can localize directly to outer or inner membrane lipids by covalent bonding due to terminal L-cysteine.
  • Lpp a major lipoprotein
  • PG peptidoglycan
  • PAL peptidoglycan-associated lipoprotein
  • Secretion proteins as a surface protein passing through outer cell membrane, has been used for the surface expression.
  • secretion proteins are not developed well and only some secretion proteins participate in passing through outer cell membrane by the proteins helping the specific secretion process.
  • a pullulanase from Klebsiella sp. is replaced with a lipid component at the N-terminus, bind to outer cell membrane and is secreted into cell media.
  • Kornacker et al. tried to express ⁇ - lactamase on cell surface by using the N-terminal fragment of pullulanase.
  • the pullulanase- ⁇ -lactamase fusion protein bound to cell surface instantly and was secreted into cell media.
  • alkaline phosphatase a periplasmic space protein
  • Ig A protease is derived from a pathogenic microorganism, Neisseria and has a unique secretion mechanism.
  • the C-terminal ⁇ -fragment of Ig A protease has a signal by which the N-terminal protease can be localized onto outer cell membrane.
  • the extruded protease After the protease reaches the outer cell membrane to be extruded on cell surface, the extruded protease is secreted into cell media by auto-hydrolysis.
  • Klauser et al. also expressed 12kDa cholera toxin B subunit stably onto cell surface [Klauser, e al., EMBOJ., 9: 1991-1999 (1990)] .
  • secretion of the fusion protein was inhibited since a protein folding is induced in periplasmic space during the secretion process.
  • flagella other cell surface structure which is present on cell surface
  • flagella a structural subunit of flagella, respective cholela toxin B subunit and other peptides derived from Hepati tis B Virus were expressed stably and these peptides could bind with the respective antibody intensively [Newton, et al., Science, 244: 70-72 (1989)] .
  • fimbrin a structural protein of fimbriae which work on cell surface like threads, foreign proteins were also expressed. As a result, only small peptides has been expressed successfully [Hedegaard, et al., Gene, 85: 115-124 (1989)] .
  • the inventors have exploited ice nucleation protein (INP) , a surface protein derived from Pseudomonas syringae KCTC 1832, as a new surface anchoring motif and developed new surface anchoring vectors containing INP and expressing foreign proteins efficiently on cell surface, a method for preparation of foreign proteins onto cell surface and use thereof.
  • INP ice nucleation protein
  • the object of the present invention is to provide surface anchoring vectors for the surface expression exploiting characteristics of ice nucleation protein (INP) .
  • the vectors of the present invention contain a secretion signal and a targeting signal which INP has in its primary sequence.
  • the object of the present invention is to provide a method for preparing foreign proteins, which uses the surface anchoring vector using characteristics of INP and expresses foreign proteins onto cell surface.
  • the present invention provides a method for preparing foreign proteins, wherein proteins are utilized conveniently even without cell disruption or isolation since the proteins are expressed onto the surface.
  • the present invention can provide uses of foreign proteins expressed onto cell surface, which comprises effective production of antibodies and antigens and production of libraries for screening antigens, binding or adsorbent proteins, physiological activators and the like.
  • foreign proteins expressed onto cell surface can be utilized to produce levan efficiently.
  • the surface anchoring vectors of the present invention can be applied to all the bacteria hosts.
  • the host can be selected among gram-negative bacteria and more preferably among Escheri chia coli , Acetohacter sp . , Pseudomonas sp . , Xanthomonas sp. , Erwinia sp. and Xymomonas sp . .
  • the pANC3 vector (accession number : KCTC 0326 BP) has been constructed, wherein central repeating domain-deleted INP gene derived from Pseudomonas syringae KCTC 1832 is contained and foreign genes can be easily cloned due to C-terminal restriction site inserted.
  • restriction sites can be inserted into the C-terminus of INP and all the surface anchoring vectors containing these restriction sites can be within the scope of the present invention.
  • the pANC3-CM2 recombinant vector has been constructed, wherein central repeating domain-deleted INP gene is contained and at the C- terminus of INP gene the N-terminus of CMCase gene are ligated to produce CMCase onto cell surface in form of fusion protein.
  • the pGINP21M vector in particular embodiments, the pGINP21M vector
  • the pASCMl recombinant vector (accession number : KCTC 0237 BP) has been constructed, wherein INP gene is contained and at the C-terminus of INP gene the N-terminus of carboxymethylcellulase (CMCase) gene are ligated to produce the CMCase onto cell surface in form of fusion protein.
  • CMCase carboxymethylcellulase
  • the pASLPl recombinant vector has been constructed, wherein INP gene is also contained and at the C-terminus of INP gene the N-terminus of a lipase gene is ligated instead of CMCase gene to produce lipase onto cell surface in form of fusion protein.
  • the pASIgl recombinant vector has been constructed, wherein INP gene is also contained and at the C-terminus of INP gene the N-terminus of single chain Fv antibody gene is ligated instead of the CMCase gene to produce single chain Fv antibody onto cell surface in form of fusion protein.
  • the pSSTS109 recombinant vector has been constructed, wherein INP gene is also contained and levansucrase gene is ligated to produce levansucrase onto cell surface in form of fusion protein.
  • Fig. 1 shows a restriction map of the pGINP21M vector which contains INP gene and some restriction sites at the C-terminus of INP gene.
  • Fig. 2 shows a restriction map of the pASCMl recombinant vector which contains CMCase gene and express CMCase onto cell surface.
  • Fig. 3 illustrates activities of CMCase expressed onto cell surface from E. coli transformant of the pASCMl recombinant vector with the graph, which compares the activities in washed cells, medium and disrupted cells respectively.
  • Fig. 4 illustrates activities of lipase expressed onto cell surface from E. coli transformant of the pASLPl recombinant vector by the degrees of lipolysis.
  • Fig. 5 illustrates binding activities of single chain Fv antibody expressed onto cell surface from E. coli transformant of the pASIgl recombinant vector with antigens by performing ELISA method.
  • Fig. 6 shows a restriction map of the pANC3 vector which contains repeating domain-deleted INP gene.
  • Fig. 7 illustrates activities of CMCase expressed onto cell surface from E. coli transformant of the pANC3- CM2 recombinant vector.
  • Fig. 8 shows immunofluorescence staining of E. coli DH5 ⁇ transformant of the pSSTS109 recombinant vector.
  • Left panels (A and C) show light microscopic images of E. coli transformant of the pZL8 vector and that of the pSSTS109 recombinant vector and right panels (B and D) show confocal fluourescent microscopic images of above E. coli transformants respectively.
  • Fig. 9 shows bioconversion of sucrose to levan in whole cell system wherein E. coli DH5 ⁇ transformant of the pSSTS109 recombinant vector is utilized.
  • Ice nucleation protein a outer cell membrane protein
  • Pseudomonas sp . , Erwinia sp . , Xanthomonas sp. and the like has a unique function which induces ice formation in supercooled water.
  • INP genes from various kinds of bacteria, especially 8 amino acid units are repeated in the central region of INP.
  • the unit is expected to provide a frame arranging the supercooled water like ice particles.
  • specific amino acids sequences are present at the N- and C-termini of INP respectively.
  • the sequences are expected to be a secretion signal and a targeting signal by which INP can pass through inner cell membrane.
  • the N- terminus of INP plays a role in binding onto outer cell membrane.
  • INP is composed of more than 1,200 amino acids and its molecular weight is 118kDa.
  • the primary amino acid sequences are composed of the N-terminal unique amino acids (15% of the total protein sequence) , the C-terminal unique amino acids (4% of the total protein sequence) and the central repeating domain (81% of the total protein sequence) .
  • 8 amino acid units are repeated exactly 122 times within the central region of INP.
  • Green et al. has investigated physiological functions of INP by using mutants of INP gene in 3 respective regions of the protein [Green, et al., Mol. Gen. Genet., 215: 165-172 (1988)] .
  • the length of the central repeating units of INP is identified to affect ice nucleation activity.
  • Lack of the repeating property reduces or loses the ice nucleation activity and only decrease in the length of the central region maintains the ice nucleation activity. Therefore, the repeating region is expected to arranges supercooled water molecules adjacent to INP for the formation of the ice particle structure regardless of protein secretion and targeting.
  • the N-terminus of INP is expected to play a role in binding onto outer cell membrane.
  • the ice nucleation activity is maintained even when the N- terminus of INP is cleaved completely. However, the C- terminal of INP is expected to play a role in secreting and targeting INP onto outer cell membrane. The ice nucleation activity reduces completely due to C-terminal cleavage.
  • INP ha ⁇ many advantages derived from its primary amino acid sequence, its structure and its characteristics.
  • INPs are expressed massively onto cell surface.
  • INP expressed onto cell surface is maintained stably in the stationery phase of cell growth.
  • INP resides on outer cell membrane and exposed to the external surface.
  • the distance between foreign protein and cell surface can be adjusted conveniently since the length of the central repeating unit of INP can be flexible according to sizes of foreign proteins.
  • INP is a stable enzyme expressed in a broad range of gram- negative bacteria, various hosts of gram-negative bacteria can be exploited for the surface expression.
  • the pGINP21 (accession number : KCTC 86089) plasmid containing requiring genes has been used.
  • a translation termination codon is excised and 3 new restriction sites, such as Bam HI, Sma I and Eco RI, are inserted by the polymerase chain reaction (PCR) , which provides insertion sites for foreign protein genes.
  • PCR polymerase chain reaction
  • the pGINP21M vector (accession number : KCTC 0239 BP) is constructed (see Fig. 1.), wherein any restriction enzyme site mentioned above is available to insert foreign protein genes by gene manipulation framing the translation codon.
  • various vectors containing all or some restriction sites at the C-terminus of INP can be constructed.
  • the surface anchoring vector constructed by the above process contains the original INP DNA sequence and expresses INP. And when foreign protein genes are ligated at the C-terminus of INP in frame, fusion proteins can be expressed and bound stably onto cell surface.
  • foreign proteins be synthesized, pass through inner cell membrane and be bound onto cell surface.
  • foreign protein genes are ligated to INP gene in frame, transforms bacteria hosts to induce for expression and the fusion protein is identified to be expressed onto cell surface.
  • the pASCMl recombinant vector (accession number : KCTC 0237 BP) has been constructed to express carboxymethylcellulase (CMCase) derived from a gram- negative bacterium, Bacillus sp . onto cell surface.
  • CMCase carboxymethylcellulase
  • the N-terminal 390bp DNA of carboxymethylcellulase gene is obtained from the pUC19 vector containing the gene [Park, et al., Enzyme Microb. Technol. , 8(12) : 725-728 (1986)] and inserted into the pGINP21M vector.
  • the C- terminal DNA fragment of CMCase is obtained from the different pUC19 vector and inserted into the vector prepared above to construct the pASCMl recombinant vector (see Fig. 2. ) .
  • E. coli is transformed with the pASCMl recombinant vector, cultured and induced for the surface expression.
  • the CMCase activity is measured by using carboxymethylcellulose as a substrate.
  • the enzyme activities of the present invention are similar in washed whole E. coli cells and disrupted E. coli cells respectively. Hence, it is expected that the total enzyme activity appears only in CMCase extruded onto cell surface.
  • carboxymethylcellulose has too high a molecular weight to penetrate outer cell membrane, the enzyme activity can appear by the process, wherein cell surface expressing CMCase is contact with a carboxymethylcellulose dissolved in substrate solution. Since cell medium does not show the CMCase activity it is suggested that CMCase be seldom separated from cell surface and limit the enzyme activity (see Fig. 3.) .
  • the pASLPl recombinant vector containing INP ha ⁇ been constructed to express lipase derived from a gram- negative bacterium, Pseudomonas sp . , onto cell surface.
  • the lipase gene is obtained from the pJH92 plasmid (Jung, Kook Hoon, Department of Biological Science, Ph D Thesis, KIST, 1990) by performing PCR and ligated with the pASCMl recombinant vector digested with Bam HI and Eco RI to construct the pASLPl recombinant vector producing the fusion protein of INP and lipase.
  • E. coli i ⁇ transformed with the pASLPl recombinant vector, cultured and induced for the surface expression. Then, the lipase activity is measured by the cupuric acetate method. Compared with host cells lacking lipase, the higher lipase activity appears in transformed E. coli expressing lipase onto cell surface. Therefore the host cells expressing lipase onto cell surface can be utilized directly in double-phase lipolysis (see the Fig. 4.) .
  • the pASIgl recombinant vector containing INP gene has been constructed to express a single chain Fv antibody.
  • the single chain Fv antibody gene is obtained from the pLUV2 plasmid which can produce the antibody as a secretion protein in E. coli and inserted by the process described above to construct the pASIgl recombinant vector.
  • E. coli is transformed, cultured and induced for the surface expression. Then, the surface expression of the single chain Fv antibody is identified by the ELISA (Enzyme-linked Immunoassay) method which measures degrees of antigens binding with surface-expressed antibodies.
  • the pSSTS109 recombinant vector acces number : KCTC 0327 BP
  • INP gene has been constructed to express levansucrase. The levansucrase gene is obtained by performing PCR with the pZL8 vector, subcloned into the pT7Blue(R) vector and then inserted into the pGINP21M vector of the present invention.
  • E. coli is transformed, cultured and induced for the surface expression.
  • the surface expression of levansucrase is observed by performing immunofluorescence staining (see
  • levansucrase expressed onto cell surface can be used to produce levan from sucrose conveniently and efficiently (see Fig. 9.) .
  • the pANC3 vector (accession number : KCTC 0326 BP) is constructed (see the Fig. 6) .
  • the pANC3-CM2 recombinant vector has been constructed to express CMCase onto cell surface. The CMCase activity was measured by the process described above (see Fig. 7.).
  • the surface expression system of the present invention has produced useful enzymes and antibodies efficiently.
  • the foreign proteins expressed above as examples illustrate the surface expression by using INP, which by no means limits the present invention. Any foreign protein can be expressed onto cell surface by cell surface expression system of the present invention.
  • INP gene the pGINP21 plasmid (7.1kb) containing INP gene cloned was used (deposit authority: Korea Research
  • SEQ. ID. No. 1 primer and a SEQ. ID. No. 2 primer were used.
  • the SEQ. ID. No. 1 primer was synthesized to insert a Kpn I restriction site and the SEQ. ID. No. 2 primer was synthesized to insert 3 restriction sites such as Bam HI, S a I and Eco RI sequentially.
  • the restriction sites inserted facilitated the subcloning of INP gene after gene amplification.
  • the gene fragment amplified by PCR machine was digested with restriction enzymes, Kpn I and Eco RI, and inserted into the pGINP21 vector already digested with Kpn I and Eco RI . As a result, the pGINP21M vector was constructed (see the Fig.
  • the size of the pGINP21M vector is 7.1kb.
  • E. coli was transformed with the pGINP21M vector of this invention and the transformed E. coli has been deposited with KRIBB, KIST on March 28, 1997 (accession number : KCTC 0239 BP) .
  • the pASCMl recombinant vector was constructed, which uses INP and can express CMCase on cell surface.
  • CMCase gene was inserted into the pGINP21M vector for the ⁇ urface expression
  • the vector was digested with Bam HI and Eco RI
  • pUC19 universal cloning vector containing the N-terminal 390bp DNA of CMCase was also digested with Bam HI and Eco RI.
  • 2 DNA fragments prepared above were joined with DNA ligase to construct the pGINP21CMl plasmid.
  • the C-terminal DNA fragment of CMCase was obtained from Eco RI sites of another pUC19 vector by digesting with Eco RI and inserted into Eco RI site of the pGINP21CMl plasmid containing the N-terminus of CMCase.
  • complete CMCase gene was joined to the C-terminus of INP gene.
  • the pASCMl recombinant vector prepared above is shown in Fig. 2. E. coli was transformed with the pASCMl recombinant vector of this invention and the transformed E. coli has been deposited with KRIBB, KIST on March 22, 1997 (accession number : KCTC 0237 BP) .
  • E. coli was transformed with the pASCMl recombinant vector, cultured in 500 ml flask containing 100 ml of LB medium and antibiotics, 100 mg/L ampicillin, and induced for the surface expression.
  • CMCase activity was measured by DNS method using carboxymethylcellulose as a substrate.
  • 1% (w/v) carboxymethylcellulose was dissolved in 50 mM citrate buffer to prepare the substrate solution.
  • 0.5 ml of this substrate solution was added, mixed well with 0.5 ml of the enzyme solution and warmed up in double boiler at 60°C for 30 minutes for reaction.
  • 3 ml of DNS solution was added, which had been prepared by dissolving 7.5 g of 2, 5-dinitrosalicilc acid, 14.0 g of NaOH, 216.1 g of Rochel salt, 5.4 g of phenol and 5.9 g of Na 2 S 2 O s sequentially in 1 L of pure water.
  • the substrate solution was reacted with DNS solution in boiling water for 5 minutes and then cooled in ice water. Using cooled solution of room temperature, absorbance at 550 nm was measured and the amount of reducing sugar released was calculated by comparing with standard curve of glucose. Enzyme 1 unit indicates the amount of enzyme which releases 1 ⁇ M glucose for 1 minute.
  • the pASLPl recombinant vector was constructed, which uses INP and can express lipase on cell surface.
  • the pASCMl recombinant vector prepared in Example 2 was digested with Bam HI and Eco RI and CMCase gene was removed to prepare the surface anchoring vector.
  • the pJH92 plasmid containing this gene was used and manipulated to insert Bam HI and Eco RI restriction sites by PCR technique.
  • the lipase gene can be obtained by digesting this pJH92 vector with Bam HI and Eco RI and was ligated into the surface anchoring vector prepared above.
  • the pASLPl recombinant vector was constructed, which can express INP and the lipase in form of fusion protein.
  • E. coli was transformed with the pASLPl recombinant vector by the calcium chloride method, cultured by the same method as described in Example 3 and induced for the surface expression. Then, the lipase activity expressed onto cell surface was measured by the cupuric acetate method as described below. 5 ml of E. coli culture fluid was mixed with 5% of olive oil substrate dissolved in 5 ml of iso-octhane and was reacted at 40°C for 1 hr. Then, the phases of solution and oil were suspended and 3 ml of the suspended solution was treated with 1 ml of the cupuric acetate reagent by shaking vigorously. Then, the absorbance at 715 nm of the reaction mixture was measured. At that time, it is expected that the higher the absorbance, the higher the enzyme activity since much oil was degraded to produce acidic lipid. As a result, the lipase activity expressed onto cell surface was measured as indicated in Fig. 4.
  • the pASIgl recombinant vector was constructed, which uses INP and can express the humanized single chain Fv antibody onto cell surface.
  • the pLUV2 plasmid which contains the single chain Fv antibody gene and can produce this antibody as a secretion protein, was digested with Sal I and Eco RI and ligated a synthetic oligonucleotide to add a new Bgl II restriction site instead of Sal I site.
  • the pASCMl recombinant vector prepared in Example 2 was digested with Eco RI and Bam HI to remove CMCase gene for the surface expression and ligated to the single chain antibody gene digested with Bgl II and Eco RI.
  • the pASIgl recombinant vector was constructed, which can expres ⁇ INP and the single chain Fv antibody in form of fusion protein.
  • E. coli was transformed with the pASIgl recombinant vector and induced by the same method as described in Example 3 for the surface expression. Then, the activity of single chain Fv antibody expressed onto cell surface was identified by ELISA method which measures degrees of antigens binding with surface-expressed antibodies. And in cells expressing antibodies and cells containing only the expression vector, the degrees of antigen-binding were compared respectively.
  • the respective cells was adjusted to have the same concentrations, harvested, washed with PBS buffer (pH 7.4) and resuspended with 1.4 ml of the same buffer. These suspension solution were divided into 25, 50, 100, 200 and 250 ⁇ l batches, then mixed with pre-S2 antigens of the critical concentration which can bind antibody
  • the overnight coated plates were blocked for 2 hours by using TBS-T buffer [0.05% (v/v) Tween-20, 10 mM Tris, pH 7.4, 0.15 M NaCl] containing 2% bovine serum albumin and washed with TBS-T buffer. Then, 10 ng of the primary antibody, H69k, was added, reacted for 2 hours and washed with the same buffer to remove unbound antibodies. And the secondary antibody bound with horse-radish peroxidase was diluted with 1000 times of the buffer and reacted. H 2 0 2 as a substrate of peroxidase and OPD as a coloring reagent were added to induce coloring and this reaction was stopped by using suphuric acid. Then absorbance at 492 nm was measured and converted into percentages against 100 % of the absorbance of the blank which does not contain cells. As a result, antigen-bindings was compared with that of control cells as indicated in Fig. 5.
  • the pSSTS109 recombinant vector was constructed, which uses INP and can express levansucrase onto cell surface.
  • ORF ORF respectively and containing new restrinction enzyme sites, Bam HI site at the beginning and Sma I and Eco RI sites at the end of ORF were used as primers .
  • the PCR products were subcloned efficiently into the pT7Blue(R) vector (Novagen Co., USA) .
  • the subcloned levansucrase gene was digested with Bam HI-Eco RI and inserted into the pGINP21M vector digested with the same enzyme.
  • the pSSTS109 recombinant vector was prepared, E. coli DH5 ⁇ was transformed and the transformed E. coli has been deposited with KRIBB, KIST on March 27, 1997 (accession number : KCTC 0327 BP) .
  • E. coli was transformed with the pSSTS109 recombinant vector and induced by the same method as described in Example 3 for the surface expression.
  • Fig. 8 The physical observation of the ⁇ urface anchored levansucrase is presented in Fig. 8. This levansucrase was stained with immunofluorescence of levansucrase- reacting antibody and FITC-labelled secondary antibody. The positive reacting cells were stained efficiently as shown in Fig. 8(D), whereas the negative reacting cells were not stained (see Fig. 8(B)) . This is a direct evidence that INP can direct foreign proteins onto outer cell membrance and be useful as a surface anchoring motif.
  • E. coli was transformed with the pANC3 vector of this invention and the transformed E. coli has been deposited with KRIBB, KIST on March 27, 1997 (accession number : KCTC 0326 BP) .
  • the pANC3-CM3 recombinant vector was constructed, which uses repeating domain-deleted INP and can express CMCase on cell surface.
  • INP is a large polypeptide with the internal repeating domain which may not be necessary to be transported onto outer cell membrane.
  • CMCase gene was also subcloned into the pANC3 anchoring vector, preparing the pANC3-CM2 recombinant vector as described in Example 12.
  • E. coli JM109 strain was transformed with the pANC3- CM2 recombinant vector and whole CMCase activity on the cell surface of the transformed E. coli JM109 was determined by performing the same procedure of Example 3. It was confirmed by measuring whole cell CMCase activity after washing the cells (see the Fig. 7) .
  • the cells grown at 42°C indicated higher level of CMCase activity than the cells grown at 37°C.
  • the pGINP21M vector and the pANC3 vector prepared in the present invention has expressed CMCase, a lipase, a humanized single chain Fv antibody, levansucrase and the like onto cell surface of E. coli efficiently.
  • This vector is very advantageous as described below.
  • the proteins expressed does not be diluted due to proteins concentration onto cell surface. And the expression can be identified easily only by washing the cells since cell disruption or protein isolation or purification is not necessary.
  • the vector of the present invention using the characteristics of INP is useful to clone foreign protein genes since the lenth of the central repeating region of INP, excepting a secretion signal and a targeting signal in the primary sequence of INP, can be flexible according to needs.
  • this vector has a outstanding advantage that the distance between the foreign protein and cell surface can be adjusted conveniently.
  • the method for the preparation of the present invention uses the characteristics of INP and can express foreign proteins stably regardless of the cell cycle. And it can be applied to various kinds of the bacteria hosts. In addition the surface expression facilitates the identification of foreign proteins on account of massive expression onto cell surface.
  • the surface expression sy ⁇ tem of the pre ⁇ ent invention can be used to produce recombinant foreign proteins efficiently, which comprises various antigens, antibodies, enzymes, binding or adsorbent proteins, peptide libraries for screening physiological activators and the like. Since this system has a broad range of applications, it can be used to produce new vaccines, anti-peptide antibodies, adsorbents for the isolation of organisms, enzymes localized onto cell surface and the like.
  • levansucrase expressed onto cell surface is very useful to produce levan from sucrose and the process of the present invention can be used for the bioconversion efficiently.
  • MOLECULAR TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO.1

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Analysing Materials By The Use Of Radiation (AREA)

Abstract

L'invention concerne des vecteurs d'ancrage superficiel, un procédé de préparation de protéines étrangères sur la surface d'une cellule et leur utilisation. Dans le procédé de l'invention, on utilise une protéine membranaire extérieure de cellule, qui est une protéine glaçogène dérivée de pseudomonas syringae, laquelle est une bactérie Gram négatif.
EP97915739A 1996-04-02 1997-04-02 Vecteur et systeme d'ancrage superficiel pour proteines etrangeres Withdrawn EP0839198A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
KR9609921 1996-04-02
KR1019960009921A KR0185335B1 (ko) 1996-04-02 1996-04-02 표면 발현 벡터 및 이를 이용한 단백질의 미생물 표면 발현 방법
PCT/KR1997/000057 WO1997037025A1 (fr) 1996-04-02 1997-04-02 Vecteur et systeme d'ancrage superficiel pour proteines etrangeres

Publications (1)

Publication Number Publication Date
EP0839198A1 true EP0839198A1 (fr) 1998-05-06

Family

ID=19454914

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97915739A Withdrawn EP0839198A1 (fr) 1996-04-02 1997-04-02 Vecteur et systeme d'ancrage superficiel pour proteines etrangeres

Country Status (9)

Country Link
US (1) US6071725A (fr)
EP (1) EP0839198A1 (fr)
JP (1) JP3285588B2 (fr)
KR (1) KR0185335B1 (fr)
CN (1) CN1117863C (fr)
AU (1) AU699792B2 (fr)
CA (1) CA2222128C (fr)
RU (1) RU2191827C2 (fr)
WO (1) WO1997037025A1 (fr)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4531899A (en) * 1998-06-25 2000-01-10 Chul-Joong Kim Surface expression system of hepatitis b virus surface antigen and core antigen of hepatitis c virus by using ice-nucleation protein
KR100312070B1 (ko) * 1999-03-15 2001-11-03 박호군 고속 선별법을 포함하는 개선된 형질을 갖는 효소 변이체의 제조방법
JP4458844B2 (ja) * 2001-08-10 2010-04-28 バイオリーダース・コーポレイション ｐｇｓＢＣＡ、ポリ−γ−グルタミン酸シンテターゼをコードする遺伝子を有する表面発現用のベクター、及びこれを用いた標的タンパク質の微生物表面発現方法
KR100462832B1 (ko) * 2002-04-11 2004-12-20 한국과학기술원 세포표면 발현용 유전자
KR100475133B1 (ko) * 2002-09-13 2005-03-10 한국생명공학연구원 효모 표면 발현 벡터를 이용하여 변이 리파제를스크리닝하는 방법 및 신규 변이 리파제
KR100690948B1 (ko) * 2004-06-17 2007-03-09 한국과학기술원 대장균의 외막 단백질을 이용한 목적 단백질의 미생물표면발현 방법
WO2012176955A1 (fr) * 2011-06-20 2012-12-27 서울대학교 산학협력단 Lipase recombinante comprenant, comme marqueur lié à son extrémité n-terminale, un acide aminé à chaîne latérale polaire chargée négativement ou un acide aminé à chaîne latérale polaire non chargée, et son procédé de production dans lequel est utilisée escherichia coli
CN105154462B (zh) * 2015-08-25 2020-06-02 大连大学 一种利用骨架蛋白Fn3在大肠杆菌体内建立N-糖基化受体蛋白模型的方法
CN105154461B (zh) * 2015-08-25 2020-05-22 大连大学 一种利用骨架蛋白Fn3在大肠杆菌体内建立N-糖基化效率检测受体蛋白模型的方法
MX2019012845A (es) * 2017-04-28 2019-11-28 Agrospheres Inc Composiciones y metodos para encapsular y administrar agroquimicos de forma escalable.
EP3615082A4 (fr) 2017-04-28 2021-05-26 Agrospheres, Inc. Compositions et procédés d'immobilisation d'enzyme
WO2019060903A1 (fr) 2017-09-25 2019-03-28 Agrospheres, Inc. Compositions et procédés de production et d'administration évolutive de produits biologiques
CN111979166B (zh) * 2020-08-14 2022-11-18 中国环境科学研究院 一种特异去除砷的工程菌及其构建方法与应用
CN112481236B (zh) * 2020-11-25 2023-06-06 武汉理工大学 一种重组蛋白INP-AidH及其制备方法和应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4978540A (en) * 1988-01-20 1990-12-18 Lee Tung Ching Production of frozen foods and other products
KR910007611A (ko) * 1989-10-14 1991-05-30 김용원 멀티 드릴링(Multi Drilling) 장치
GB8923998D0 (en) * 1989-10-25 1989-12-13 Nestle Sa Food additives
JP3094485B2 (ja) * 1991-03-27 2000-10-03 株式会社島津製作所 植物形質転換体を検出するためのオリゴヌクレオチドおよびその検出方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9737025A1 *

Also Published As

Publication number Publication date
CN1117863C (zh) 2003-08-13
JPH10508207A (ja) 1998-08-18
AU699792B2 (en) 1998-12-17
RU2191827C2 (ru) 2002-10-27
KR970070205A (ko) 1997-11-07
CA2222128A1 (fr) 1997-10-09
CN1185809A (zh) 1998-06-24
KR0185335B1 (ko) 1999-04-01
AU2308797A (en) 1997-10-22
JP3285588B2 (ja) 2002-05-27
US6071725A (en) 2000-06-06
WO1997037025A1 (fr) 1997-10-09
CA2222128C (fr) 2003-06-17

Similar Documents

Publication Publication Date Title
JP4458844B2 (ja) ｐｇｓＢＣＡ、ポリ−γ−グルタミン酸シンテターゼをコードする遺伝子を有する表面発現用のベクター、及びこれを用いた標的タンパク質の微生物表面発現方法
EP0746621B1 (fr) Expression de proteines sur la surface de bacteries
AU699792B2 (en) Surface anchoring vector and system for foreign proteins thereof
KR100522398B1 (ko) 포자 표면발현 방법
EP0584167B1 (fr) Adn recombine codant pour un peptide signal, polypeptide d'interaction et sequence d'ancrage de membrane
KR100395307B1 (ko) 효모로부터 분리한 신규 세포벽 부착 매개 단백질, 그유전자 및 이를 이용한 세포 표면 발현 시스템
CN113603756B (zh) 谷氨酸棒杆菌膜蛋白Ncgl2775及其表面展示系统和构建方法
KR100518953B1 (ko) 포자 외막단백질을 이용한 목적단백질의 표면발현 방법
KR100810527B1 (ko) 유전자 담체 표면 전시방법
CN113801211A (zh) 谷氨酸棒杆菌蛋白Ncgl0717及其表面展示系统和构建方法
JP7116809B2 (ja) バチルス属菌株由来のポリ－γ－グルタミン酸合成遺伝子を用いた表面発現ベクター、及びそれを用いたタンパク質の微生物表面発現方法
KR20050119786A (ko) 대장균의 외막 단백질을 이용한 목적 단백질의 미생물표면발현 방법
US7129060B1 (en) Export systems for recombinant proteins
JPH11514201A (ja) 安定な融合タンパク質の産生に使用する細菌および該細菌の検出方法
CN113603755A (zh) 谷氨酸棒杆菌蛋白Ncgl1307及其表面展示系统和构建方法
Kuen et al. Analysis of S-layer proteins and genes
JP2022542883A (ja) 緑膿菌のＯｐｒＦタンパク質に対する抗体、薬剤としてのその使用、及び前記抗体を含む医薬組成物
JP2021029171A (ja) ブレビバチルス菌を用いた表層提示法
JPH04349888A (ja) マルトテトラオース生成酵素遺伝子を含有するプラスミド及び該プラスミドを有する微生物並びに該微生物を用いるマルトテトラオース生成酵素の製造法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19980224

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): DE DK FR GB NL SE

17Q First examination report despatched

Effective date: 20020923

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040702